Phosphatases are enzymes that remove phosphate from a molecule. The removal of phosphate is called dephosphorylation. Kinases are enzymes that catalyze the addition of phosphate to a molecule. The addition of phosphate by a kinase is called phosphorylation. When the kinase substrate is a protein molecule, the amino acids commonly phosphorylated are serine, threonine and tyrosine. Kinases and phosphatases often represent competing forces within a cell to transmit, attenuate, or otherwise modulate cellular signals and cellular control mechanisms. Kinases and phosphatases have both overlapping and unique natural substrates. Cellular signals and control mechanisms, as regulated by kinases, phosphatases, and their natural substrates are a target of research tool design and drug design.
PP1 or protein phosphatase 1 is involved in regulation of numerous cellular processes, including heart function. Functional inactivation of the naturally occurring PP1 inhibitor 1 (INH-1) was shown to be associated with the heart failure in human patients (El-Armouche et al. 2004 Cardiovasc Res. 61:87-93). Inactivation of PP1 inhibitor results in the increased activity of PP1. Consistent with this notion, studies (Yamada et al. 2006 FASEB J 20:1197-9) showed that adenovirus-mediated expression of the naturally occurring inhibitor, INH-2, alleviated progression of heart failure and improved survival of the animals. Regulation of cardiac function by PP1 may occur via PP1 mediated dephosphorylation of phospholamban, which in turn regulates the activity of the calcium pump SERCA2 in the sarco(endo)plasmic reticulum (SR) of the muscle cells (Neumann 2002 Basic Res Cardiol. 97 Suppl 1:191-5). Additionally, transgenic animals over-expressing INH-2 had improved heart function and increased levels of phosphorylated PLB (Kirchhefer et al. 2005 Cardiovasc Res 68:98-108).
Mammalian Protein Phosphatase 1 is also known as PP1. The enzymatic activity of PP1 catalytic subunit has been studied (see Terrak et al. 2004 Nature 429:780-4). Unlike kinases, which have more distinct substrate specificites, PP1 catalytic subunit is capable of dephosphorylating many phospho-proteins. It is currently accepted that phosphatase substrate specificity is determined largely by phosphatase binding partners, non-catalytic subunits, and/or regulatory subunits (see for example, Cohen J Cell Sci 115:241-256, 2002; Ceulemans et al. Physiol Rev 84:1-39, 2004).
Numerous phosphoprotein substrates for PP1 have been identified, including, phospholamban, aurora beta kinase, TP53, MYPT1, RNA polymerase II, PPP1R3C, retinoblastoma, MST1R, cdc25, EF2, BAD, BRCA, histoneH2AX, and IP3 receptor. Furthermore, several cellular protein regulators of PP1 have been identified, including, scapinin, PNUTS, PPPIRA, neurabinI, NIPP1, CPI17, DARPP32, neurabinII, and PPPIR2. Some PP1 substrates, regulators and other research on PP1 biology are described in the following references: Ammosova et al. 2005 Retrovirology 2:47; Ayllon et al. 2000 EMBO J. 19:2237-2246; Carr et al. 2002 Mol. Cell Biol. 22:4124-4135; Egloff et al. 1997 EMBO J. 16:1876-1887; Eto et al. 2004 PNAS 101:8888-8893; Jideama et al. 2006 Int. J. Biol Sci. 2:1-9; Lees-Miller et al. 1991 Mol. Cell Biol. 12:5041-5049; Li et al. 2006 Oncogene 25:3006-3022; Liu et al. 2005 Eur. J. Neurosci. 22:1942-1950; Liu et al. 2002 Cancer Res. 62:6357-6361; Margolis et al. 2003 EMBO J. 22:5734-5745; Champion 2005 Circ. Res. 96:708-710; Ohki et al. 2003 J Mol Biol 326:1539-47; Pathak et al. 2005 Circ. Res. 96:756-766; Quevedo et al. 2003 J. Biol. Chem. 278:16579-16586; Rubin et al. 2001 Oncogene 20:3776-3785; Santoro et al. 2003 Biochem. J. 376:587-594; Shmueli et al. 2006 Mol. Cell. Neurosci. 32:15-26; Strack et al. 1997 J. Neurochem. 68:2119-2128; Szatmari et al. 2005 J. Biol. Chem. 280:37526-37535; Tang et al. 2003 J. Neurosci. 23:403-415; Tsukada et al. 2006 BBRC 343:839-847; Uematsu et al. 2005 J. Neurochem. 95:1642-1652; Walter et al. 2000 Oncogene 19:4906-4916; Washington et al. 2002 J. Biol. Chem. 277:40442-40448; Yamada et al. 2006 FASEB J. 20:1197-9; Zhan et al. 2003 J. Immunology 171:3785-3793; Siino et al. 2002 BBRC 297:1318-1323; Welsh et al. 1997 Analyt. Biochem. 244:16-21; Rameau et al. 2004 J. Biol. Chem. 279:14307-14314; Toyoshima et al. 2003 PNAS 100:467-472; Toyofuko et al. 1994 J. Biol. Chem. 269:22929-22932; Hagiwara et al. 1992 Cell 70: 105-113; Ji et al. 2003 J. Biol. Chem. 278:25063-25071; Kimura et al. 1998 FEBS Letters 425:509-512; Kimura et al. 1997 J. Biol. Chem. 272:15061-15064; Kimura et al. 1996 J. Biol. Chem. 271:21726-21731; Haghighi et al. 2001 J. Biol. Chem. 276:24145-24152; Jin et al. 2003 J. Biol. Chem. 28:30677; Beullens et al. 2000 Biochem. J. 352:651; Sagara et al. 2003 J. Biol. Chem. 278:45611; Bibb et al. 1999 Nature 402:669; Huang et al. 1999 J. Biol. Chem. 274:7870; Landsverk et al. 2005 Biochem. J. 390:709; Kim et al. 2003 J. Biol. Chem. 278:13819; Endo et al. 1996 Biochemistry 35:5220; Weiser et al. 2004 J. Biol. Chem. 279:48904; Park et al. 1994 J. Biol. Chem. 269:944; Oliver et al. 2002 Mol. Cell. Biol. 13:4690; Yamawaki et al. 2001 BBRC 285:1040-1045; Deng et al. 2002 Biochem. J. 36:17
Small molecule inhibitors of PP1 are known in the art and include okadaic acid and microstatin. Furthermore, kits for assaying PP1 activity are available commercially, such as those available from Sigma-Aldrich (St. Louis, Mo.), New England Biolabs (Ipswich, Mass.), and Promega (Madison, Wis.).
Description of Polypeptide and Polynucleotide Sequences
SEQ ID NOS:1-56 are examples of polyligands and polynucleotides encoding them.
Specifically, the PP1 polyligand of SEQ ID NO:1 is encoded by SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:3 and SEQ ID NO:4 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:1 is an embodiment of a polyligand of the structure A-S1-B-S2-C, wherein A is SEQ ID NO:92 (wherein Xaa is Ala), B is SEQ ID NO:68 (wherein Xaa is Glu), C is SEQ ID NO:93 (wherein Xaa is Glu), and wherein S1 is a spacer of the amino acid sequence PGAGG (SEQ ID NO: 269) and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure A-S1-B-S2-C is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:5 is encoded by SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:7 and SEQ ID NO:8 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:5 is an embodiment of a polyligand of the structure A-S1-B-S2-C, wherein A is SEQ ID NO:95, B is SEQ ID NO:69 (wherein Xaa is Ser), C is SEQ ID NO:96 (wherein Xaa is Thr), and wherein S1 is a spacer of the amino acid sequence PGAGG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure A-S1-B-S2-C is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:9 is encoded by SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:11 and SEQ ID NO:12 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:9 is an embodiment of a polyligand of the structure X-S3-Y-S2-Z, wherein X is SEQ ID NO:102 (wherein Xaa is Ser), Y is SEQ ID NO:72 (wherein Xaa is Asp), Z is SEQ ID NO:100 (wherein Xaa is Thr), and wherein S3 is a spacer of the amino acid sequence PGGAG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270. A polyligand of structure X-S3-Y-S2-Z is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:13 is encoded by SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:15 and SEQ ID NO:16 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:13 is an embodiment of a polyligand of the structure X-S3-Y-S2-Z, wherein X is SEQ ID NO:101, Y is SEQ ID NO:74 (wherein Xaa is Asp), Z is SEQ ID NO:79 (wherein Xaa is Ser or Thr), and wherein S3 is a spacer of the amino acid sequence PGGAG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure X-S3-Y-S2-Z is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:17 is encoded by SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:19 and SEQ ID NO:20 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:17 is an embodiment of a polyligand of the structure X-S4-Y-S2-Z, wherein X is SEQ ID NO:93 (wherein Xaa is Glu), Y is SEQ ID NO:75 (wherein Xaa is Ser or Asp), Z is SEQ ID NO:92 (wherein Xaa is Ala), and wherein S4 is a spacer of the amino acid sequence PPGAG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure X-S4-Y-S2-Z is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:21 is encoded by SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:23 and SEQ ID NO:24 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:21 is an embodiment of a polyligand of the structure D-S3-E-S2-F, wherein D is SEQ ID NO:73 (wherein Xaa is Asp), E is SEQ ID NO:95, F is SEQ ID NO:98 (wherein Xaa is Glu or Thr), and wherein S3 is a spacer of the amino acid sequence PGGAG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure D-S3-E-S2-F is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:25 is encoded by SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:27 and SEQ ID NO:28 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:25 is an embodiment of a polyligand of the structure D-S3-E-S2-F, wherein D is SEQ ID NO:101, E is SEQ ID NO:79 (wherein Xaa is Ser or Thr), F is SEQ ID NO:108 (wherein Xaa is Glu or Asp), and wherein S3 is a spacer of the amino acid sequence PGGAG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure D-S3-E-S2-F is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:29 is encoded by SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:31 and SEQ ID NO:32 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:29 is an embodiment of a polyligand of the structure D-S3-E-S2-F, wherein D is SEQ ID NO:96 (wherein Xaa is Thr), E is SEQ ID NO:69 (wherein Xaa is Ser or Thr), F is SEQ ID NO:95, and wherein S3 is a spacer of the amino acid sequence PGGAG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure D-S3-E-S2-F is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:33 is encoded by SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:35 and SEQ ID NO:36 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:33 is an embodiment of a polyligand of the structure H-S3-J-S2-K, wherein H is SEQ ID NO:80 (wherein Xaa is Asp or Ser or Thr), J is SEQ ID NO:109 (wherein Xaa is Glu), K is SEQ ID NO:110 (wherein Xaa is Glu), and wherein S3 is a spacer of the amino acid sequence PGGAG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure H-S3-J-S2-K is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:37 is encoded by SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:39 and SEQ ID NO:40 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:37 is an embodiment of a polyligand of the structure H-S3-J-S2-K, wherein H is SEQ ID NO:80 (wherein Xaa is Glu or Ser or Thr), J is SEQ ID NO:109 (wherein Xaa is Glu), K is SEQ ID NO:110 (wherein Xaa is Glu), and wherein S3 is a spacer of the amino acid sequence PGGAG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure H-S3-J-S2-K is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:41 is encoded by SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:43 and SEQ ID NO:44 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:41 is an embodiment of a polyligand of the structure A-S5-B-S2-C, wherein A is SEQ ID NO:92 (wherein Xaa is Ser or Thr), B is SEQ ID NO:68 (wherein Xaa is Glu), C is SEQ ID NO:93 (wherein Xaa is Glu), and wherein S5 is a spacer of the amino acid sequence PGAGG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure A-S5-B-S2-C is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:45 is encoded by SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:47 and SEQ ID NO:48 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:45 is an embodiment of a polyligand of the structure A-S5-B-S2-C, wherein A is SEQ ID NO:93 (wherein Xaa is Glu), B is SEQ ID NO:82 (wherein Xaa is Glu), C is SEQ ID NO:111 (wherein Xaa is Ala), and wherein S5 is a spacer of the amino acid sequence PGAGG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure A-S5-B-S2-C is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:49 is encoded by SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:51 and SEQ ID NO:52 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:49 is an embodiment of a polyligand of the structure A-S5-B-S2-C, wherein A is SEQ ID NO:105 (wherein Xaa is Glu), B is SEQ ID NO:76 (wherein Xaa is Ser), and wherein S5 is a spacer of the amino acid sequence PGAGG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure A-S5-B-S2-C is also called herein a heteropolyligand, shown generically in FIG. 4G.
The PP1 polyligand of SEQ ID NO:53 is encoded by SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:55 and SEQ ID NO:56 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:53 is an embodiment of a polyligand of the structure A-S5-B-S2-C, wherein A is SEQ ID NO:108 (wherein Xaa is Asp or Glu or Ala), B is SEQ ID NO:67 (wherein Xaa is Glu), C is SEQ ID NO:100 (wherein Xaa is Glu), and wherein S5 is a spacer of the amino acid sequence PGAGG (SEQ ID NO: 269), and S2 is a spacer of amino acid sequence PGAAG (SEQ ID NO: 270). A polyligand of structure A-S5-B-S2-C is also called herein a heteropolyligand, shown generically in FIG. 4G.
SEQ ID NOS:57-66 and SEQ ID NOS:83-91 are full length PP1 protein substrates and regulators, respectively. These sequences have the following public database accession numbers: NP—054829, NP—003591, NP—150281, BAC82348, NP—000537, NP—006732, NP—115984, NP—002471, Q9UD71, NP—002705, NP—000928, NP—005389, NP—000312, NP—002438, NP—002658, NP—006232, NP—060120, NP—001952, and NP—001781. Each of the sequences represented by these accession numbers is incorporated by reference herein. In SEQ ID NOS:57-66, the positions of the amino acid(s) dephosphorylatable by PP1 are represented by Xaa. In a parental wild type reference sequence, Xaa is serine or threonine. In the ligands of the invention, Xaa is any amino acid. In some embodiments Xaa is alanine. Furthermore, in SEQ ID NOS:83-91, the positions of the amino acid(s) modified from a parental wild type reference sequence are represented by Xaa. In the ligands of the invention, Xaa is any amino acid. In some embodiments, Xaa is aspartate and/or glutamate.
SEQ ID NOS:67-82 are partial sequences of SEQ ID NOS:57-66, which represent examples of polypeptide ligand sequences where the location(s) of the PP1 dephosphorylatable serine or threonine in the natural parental polypeptide is designated as Xaa.
SEQ ID NOS:92-111 are partial sequences of SEQ ID NOS:83-91, which represent examples of peptide ligand sequences where the location(s) of amino acid(s) modified from a parental wild type reference sequence are designated as Xaa.
Amino acid sequences containing Xaa encompass polypeptides where Xaa is any amino acid.
Below is a descriptive annotation of several heteropolyligand embodiments of the invention.
SEQ ID NO:1 Partial NIPP1-spacer-Partial Aurora beta Kinase-spacer-Partial CPI17
SEQ ID NO:5 Partial Scapinin-spacer-Partial TP53-spacer-Partial PPP1R1A
SEQ ID NO:9 Partial NeurabinII-spacer-Partial MYPT1-spacer-Partial DARPP32
SEQ ID NO:13 Partial PNUTS-spacer-Partial RNAPII-spacer-Partial PTG
SEQ ID NO:17 Partial CPI17-spacer-Partial Rb-spacer-Partial NIPP1
SEQ ID NO:21 Partial MST1R-spacer-Partial Scapinin-spacer-Partial DARPP32
SEQ ID NO:25 Partial PNUTS-spacer-Partial PTG-spacer-Partial PPPIR1A
SEQ ID NO:29 Partial PPPIR1A-spacer-Partial TP53-spacer-Partial Scapinin
SEQ ID NO:33 Partial PLN-spacer-Partial PPPIR1A-spacer-Partial PPPIR2
SEQ ID NO:37 Partial PLN-spacer-Partial PPPIR1A-spacer-Partial PPPIR2
SEQ ID NO:41 Partial NIPP1-spacer-Partial Aurora beta kinase-spacer-Partial CPI17
SEQ ID NO:45 Partial CPI17-spacer-Partial RNAPII-spacer-Partial NIPPI
SEQ ID NO:49 Partial PPPIR2-spacer-Partial CDC25-spacer-Partial NeurabinI
SEQ ID NO:53 Partial PPPIR1A-spacer-Partial EF2-spacer-Partial DARPP32
SEQ ID NO:112 is another example of a full length PP1 substrate, human phospholamban, database accession NP—002658.
SEQ ID NOS:113-136 are monomeric PP1 ligands, wherein Xaa at positions 16 or 17 are amino acids other than serine or threonine; and wherein X at other positions is an amino acid other than found in the corresponding position of SEQ ID NO:112.
SEQ ID NOS:137-156 are monomeric PP1 ligands, wherein Xaa at positions 16 or 17 are amino acids other than serine or threonine.
SEQ ID NOS:157-160 are monomeric PP1 ligands, wherein Xaa at positions 7 or 8 are amino acids other than serine or threonine.
SEQ ID NOS:161-164 are monomeric PP1 ligands, wherein Xaa at positions 6 or 7 are amino acids other than serine or threonine.
SEQ ID NOS:165-176 are further examples of polyligands and polynucleotides encoding them.
Specifically, the PP1 polyligand of SEQ ID NO:165 is encoded by SEQ ID NO:166, SEQ ID NO:167 and by SEQ ID NO:168, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:167 and SEQ ID NO:168 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:165 is an embodiment of a polyligand of the structure A-S1-B, wherein A is SEQ ID NO:139 (wherein Xaa is Asp, Glu, or Ala), B is SEQ ID NO:136 (wherein Xaa is Asp, Glu, or Ala), and wherein S1 is a spacer of the amino acid sequence GGGG (SEQ ID NO: 271). A polyligand of structure A-S1-B is also called herein a heteropolyligand, shown generically in FIG. 4A.
The PP1 polyligand of SEQ ID NO:169 is encoded by SEQ ID NO:170, SEQ ID NO:171 and by SEQ ID NO:172, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:171 and SEQ ID NO:172 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:165 is an embodiment of a polyligand of the structure A-S1-B, wherein A is SEQ ID NO:152 (wherein Xaa is Asp, Glu, or Ala), B is SEQ ID NO:136 (wherein Xaa is Asp, Glu, or Ala), and wherein S1 is a spacer of the amino acid sequence GGGG (SEQ ID NO: 271). A polyligand of structure A-S1-B is also called herein a hete opolyligand, shown generically in FIG. 4A.
The PP1 polyligand of SEQ ID NO:173 is encoded by SEQ ID NO:174, SEQ ID NO:175 and by SEQ ID NO:176, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:175 and SEQ ID NO:176 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:173 is an embodiment of a polyligand of the structure A-S1-B-S1-C-S1-D, wherein A is SEQ ID NO:145 (wherein Xaa is Asp or Glu), B is SEQ ID NO:157 (wherein Xaa is Asp or Glu), C is SEQ ID NO:161 (wherein Xaa is Asp or Glu), D is SEQ ID NO:164 (wherein Xaa is Asp or Glu), and wherein S1 is a spacer of the amino acid sequence GGGG (SEQ ID NO:271). A polyligand of structure A-S1-B-S1-C-S1-D is also called herein a heteropolyligand, shown generically in FIG. 4D.
In SEQ ID NOS:113-164, the positions of the amino acid(s) dephosphorylatable by PP1 correspond to positions 16 and 17 of SEQ ID NO:112 and are represented by Xaa. In wild-type proteins at positions corresponding to amino acid 16 and 17, Xaa is serine or threonine. In the ligands of the invention, Xaa is any amino acid. In some embodiments, Xaa is other than serine or threonine. In other embodiments, Xaa is Glu, Asp, or Ala.
SEQ ID NOS:113-136 represent examples of monomeric peptide ligand sequences containing a sarco(endo)plasmic reticulum localization signal at the C-terminal.
SEQ ID NOS:137-164 represent examples of monomeric peptide ligand sequences lacking a specific localization signal.
SEQ ID NOS:177-200 are further examples of polyligands and polynucleotides encoding them.
Specifically, the PP1 polyligand of SEQ ID NO:177 is encoded by SEQ ID NO:178, SEQ ID NO:179, and SEQ ID NO:180, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:179 and SEQ ID NO:180 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:177 is an embodiment of a polyligand of the structure A-B, wherein A is SEQ ID NO:215 (wherein Xaa is Asp) and B is SEQ ID NO:217 (wherein Xaa is Asp). A polyligand of structure A-B is also called herein a heteropolyligand, shown generically in FIG. 3A.
The PP1 polyligand of SEQ ID NO:181 is encoded by SEQ ID NO:182, SEQ ID NO:183, and SEQ ID NO:184, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:183 and SEQ ID NO:184 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:181 is an embodiment of a polyligand of the structure A-B, wherein A is SEQ ID NO:214 (wherein Xaa is Glu) and B is SEQ ID NO:217 (wherein Xaa is Asp). A polyligand of structure A-B is also called herein a heteropolyligand, shown generically in FIG. 3A.
The PP1 polyligand of SEQ ID NO:185 is encoded by SEQ ID NO:186, SEQ ID NO:187, and SEQ ID NO:188, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:187 and SEQ ID NO:188 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:185 is an embodiment of a polyligand of the structure A-B, wherein A is SEQ ID NO:216 (wherein Xaa is Glu) and B is SEQ ID NO:218 (wherein Xaa is Glu). A polyligand of structure A-B is also called herein a heteropolyligand, shown generically in FIG. 3A.
The PP1 polyligand of SEQ ID NO:189 is encoded by SEQ ID NO:190, SEQ ID NO:191, and SEQ ID NO:192, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:191 and SEQ ID NO:192 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:189 is an embodiment of a polyligand of the structure A-B, wherein A is SEQ ID NO:212 (wherein Xaa is Asp) and B is SEQ ID NO:213 (wherein Xaa is Asp). A polyligand of structure A-B is also called herein a heteropolyligand, shown generically in FIG. 3A.
The PP1 polyligand of SEQ ID NO:193 is encoded by SEQ ID NO:194, SEQ ID NO:195, and SEQ ID NO:196, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:195 and SEQ ID NO:196 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:193 is an embodiment of a polyligand of the structure A-B, wherein A is SEQ ID NO:223 (wherein Xaa is Ala) and B is SEQ ID NO:222 (wherein Xaa is Ala). A polyligand of structure A-B is also called herein a heteropolyligand, shown generically in FIG. 3A.
The PP1 polyligand of SEQ ID NO:197 is encoded by SEQ ID NO:198, SEQ ID NO:199, and SEQ ID NO:200, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:199 and SEQ ID NO:200 contain alternative flanking restriction sites applicable to modular cloning methods. SEQ ID NO:197 is an embodiment of a polyligand of the structure A-B, wherein A is SEQ ID NO:221 and B is SEQ ID NO:220. A polyligand of structure A-B is also called herein a heteropolyligand, shown generically in FIG. 3A.
SEQ ID NOS:201-207 and SEQ ID NOS:208-211 are full length PP1 protein substrates and regulators, respectively. These sequences have the following public database accession numbers: NP—003591, BAC82348, NP—006732, NP—002471, NP—002705, NP—060120, NP—004313, NP—620221, NP—002096, NP—001007236, and NP—009225. Each of the sequences represented by these accession numbers is incorporated by reference herein. In SEQ ID NOS:201-207 the positions of the amino acid(s) dephosphorylatable by PP1 are represented by Xaa. In a parental wild type reference sequence, Xaa is serine or threonine. In the ligands of the invention, Xaa is any amino acid. In some embodiments, Xaa is alanine. In other embodiments of the invention, Xaa is aspartate and/or glutamate.
Furthermore, in SEQ ID NOS:208-211, the positions of the amino acid(s) modified from a parental wild type reference sequence are represented by Xaa. In the ligands of the invention, Xaa is any amino acid. In some embodiments, Xaa is aspartate and/or glutamate.
SEQ ID NOS:212-219 are partial sequences of SEQ ID NOS:201-207, which represent examples of polypeptide ligand sequences where the location(s) of the PP1 dephosphorylatable serine or threonine in the natural parental polypeptide is designated as Xaa.
SEQ ID NOS:220-223 are partial sequences of SEQ ID NOS:208-211, which represent examples of peptide ligand sequences where the location(s) of amino acid(s) modified from a parental wild type reference sequence are designated as Xaa.
SEQ ID NOS:212-223 are further examples of monomeric PP1 ligands.
Amino acid sequences containing Xaa encompass polypeptides where Xaa is any amino acid.
SEQ ID NO:224-250 are further examples of polyligands and polynucleotides encoding them.
Specifically, the PP1 polyligand of SEQ ID NO:224 is encoded by SEQ ID NO:225 and SEQ ID NO:226, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:226 contains flanking restriction sites applicable to modular cloning methods. SEQ ID NO:224 is an embodiment of a polyligand of the structure A-S-B-C, wherein A is SEQ ID NO:251, B is SEQ ID NO:256, C is SEQ ID NO:262, and S is a spacer of SEQ ID NO:263. A polyligand of structure A-S-B-C is also called herein a heteropolyligand, shown generically in FIG. 4E.
The PP1 polyligand of SEQ ID NO:227 is encoded by SEQ ID NO:228 and SEQ ID NO:229, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:229 contains flanking restriction sites applicable to modular cloning methods. SEQ ID NO:227 is an embodiment of a polyligand of the structure A-S-B-C, wherein A is SEQ ID NO:252, B is SEQ ID NO:256, C is SEQ ID NO:262, and S is a spacer of SEQ ID NO:263. A polyligand of structure A-S-B-C is also called herein a heteropolyligand, shown generically in FIG. 4E.
The PP1 polyligand of SEQ ID NO:230 is encoded by SEQ ID NO:231 and SEQ ID NO:232, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:232 contains flanking restriction sites applicable to modular cloning methods. SEQ ID NO:230 is an embodiment of a polyligand of the structure A-B-C, wherein A is SEQ ID NO:257, B is SEQ ID NO:262 and C is SEQ ID NO:253. A polyligand of structure A-B-C is also called herein a heteropolyligand, shown generically in FIG. 3B.
The PP1 polyligand of SEQ ID NO:233 is encoded by SEQ ID NO:234 and SEQ ID NO:235, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:235 contains flanking restriction sites applicable to modular cloning methods. SEQ ID NO:233 is an embodiment of a polyligand of the structure A-B-C, wherein A is SEQ ID NO:257, B is SEQ ID NO:262 and C is SEQ ID NO:254. A polyligand of structure A-B-C is also called herein a heteropolyligand, shown generically in FIG. 3B.
The PP1 polyligand of SEQ ID NO:236 is encoded by SEQ ID NO:237 and SEQ ID NO:238, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:238 contains flanking restriction sites applicable to modular cloning methods. SEQ ID NO:236 is an embodiment of a polyligand of the structure A-S1-B-S2, wherein A is SEQ ID NO:252, B is SEQ ID NO:261, S1 is a spacer of SEQ ID NO:264, and S2 is a spacer of SEQ ID NO:265. A polyligand of structure A-S1-B-S2 is also called herein a heteropolyligand, shown generically in FIG. 4B.
The PP1 polyligand of SEQ ID NO:239 is encoded by SEQ ID NO:240 and SEQ ID NO:241, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:241 contains flanking restriction sites applicable to modular cloning methods. SEQ ID NO:239 is an embodiment of a polyligand of the structure A-S1-B-S2, wherein A is SEQ ID NO:255, B is SEQ ID NO:258, S1 is a spacer of SEQ ID NO:264, and S2 is a spacer of SEQ ID NO:265. A polyligand of structure A-S1-B-S2 is also called herein a heteropolyligand, shown generically in FIG. 4B.
The PP1 polyligand of SEQ ID NO:242 is encoded by SEQ ID NO:243 and SEQ ID NO:244, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:244 contains flanking restriction sites applicable to modular cloning methods. SEQ ID NO:242 is an embodiment of a polyligand of the structure A-S1-B-S2, wherein A is SEQ ID NO:255, B is SEQ ID NO:259, S1 is a spacer of SEQ ID NO:264, and S2 is a spacer of SEQ ID NO:265. A polyligand of structure A-S1-B-S2 is also called herein a heteropolyligand, shown generically in FIG. 4B.
The PP1 polyligand of SEQ ID NO:245 is encoded by SEQ ID NO:246 and SEQ ID NO:247, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:247 contains flanking restriction sites applicable to modular cloning methods. SEQ ID NO:245 is an embodiment of a polyligand of the structure A-B-C, wherein A is SEQ ID NO:256, B is SEQ ID NO:262 and C is SEQ ID NO:254. A polyligand of structure A-B-C is also called herein a heteropolyligand, shown generically in FIG. 3B.
The PP1 polyligand of SEQ ID NO:248 is encoded by SEQ ID NO:249 and SEQ ID NO:250, wherein codons are optimized for mammalian expression and vector insertion, and wherein SEQ ID NO:250 contains flanking restriction sites applicable to modular cloning methods. SEQ ID NO:248 is an embodiment of a polyligand of the structure A-S1-B-S2, wherein A is SEQ ID NO:255, B is SEQ ID NO:261, S1 is a spacer of SEQ ID NO:264, and S2 is a spacer of SEQ ID NO:265. A polyligand of structure A-S1-B-S2 is also called herein a heteropolyligand, shown generically in FIG. 4B.
SEQ ID NO:266-268 are full length PP1 catalytic subunit binding proteins. These sequences have the following public database accession numbers: NP—990454.1, NP—150281.1, NP—001082695.1. Each of the sequences represented by these accession numbers is incorporated by reference herein.
In one embodiment of the invention, where truncation fragments of SEQ ID NO:267 are utilized, amino acids corresponding to positions 38, 66 and 67 of SEQ ID NO:267 may be optionally mutated to any amino acid. In one embodiment, a mutation may generate a pseudophosphorylated polypeptide, such as by replacing with an acidic amino acid such as aspartate or glutamate. In a specific embodiment, the mutations are T38D and/or G66E and/or M67A.
In another embodiment of the invention, where fragments of SEQ ID NO:268 are utilized, amino acids corresponding to positions 35 and 38 of SEQ ID NO:268 may be optionally mutated to any amino acid. In one embodiment, a mutation may generate a pseudophosphorylated polypeptide, such as by replacing with an acidic amino acid such as aspartate or glutamate. In a specific embodiment, the mutations are T35D and/or T38D.
SEQ ID NOS:251-258 are partial sequences (truncation fragments) of SEQ ID NO:266-268.
SEQ ID NOS:259-262 are mutated partial sequences (mutated truncation fragments) of SEQ ID NOS:267-268.
SEQ ID NOS:263-265 are short peptide spacer amino acid sequences.
SEQ ID NOS:251-262 are further examples of monomeric PP1 ligands.
Three letter amino acid codes and one letter amino acid codes are used herein as is commonly known in the art.